Method for treating arthritis or inflammation with Il-1α or derivatives thereof

ABSTRACT

Interleukin-1 alpha  (IL-1- alpha ) and derivatives, and their pharmaceutical compositions for use in treating arthritis or inflammation.

This is a divisional of application Ser. No. 07/023,373, filed Mar. 9,1987, now U.S. Pat. No. 5,008,374.

The present invention relates to novel polypeptides, and moreparticularly to novel derivatives of interleukin-1α (IL-1α) and to themedicinal use of IL-1α and the novel derivatives thereof.

BACKGROUND OF THE INVENTION

The Second International Lymphokine Workship decided to adopt a unifiedname, interleukin-1 (IL-1), for the physiologically active substanceswhich had been referred to as lymphocyte activating factor (LAF),mitogenic protein, helper peak-1, T-cell replacing factor III (TRF-III),T-cell replacing factor Mφ (TRFM), B-cell activating factor, B-celldifferentiation factor, etc. (Cellular Immunol., 48, 433-436 (1979)).This decision is based on the reason that these physiologically activesubstances can not be distinguished from one another as differentsubstances but are expressed variously merely with reference tophysiological activities as interpreted from different angles.

Further it is reported that IL-1 activates T lymphocytes and Blymphocytes, has activity to promote production of interleukin-2 andantibodies, acts on liver tissues to promote protein synthesis andpossesses activity to promote production of prostaglandins (see Reviewsof Infectious Disease, Vol. 6, No. 1, 51-59 (1984), New England J. ofMed., 311, 1413 (1984), etc.).

Whereas IL-1 itself still remains to be clarified as a substance, it isonly recently that reports are made on the presence of genes coding forpolypeptides having LAF activity or precursor thereof (Proc. Natl. Acad.Sci., Vol. 81, 7907-7911 (1984), Nature, Vol. 315, (1985), Nucleic AcidResearch, Vol. 13 (16), 5869 (1985)).

According to the report, a culture supernatant obtained by generecombination techniques was found to have LAF activity, and based onthis finding, it is speculated that the polypeptide represented by thefollowing formula (A) is a polypeptide having LAF activity and is termed"IL-1α." ##STR1##

However, no reports have been made to the effect that the above activesubstance, as well as the physiologically active substance which isreportedly known as the so-called IL-1, is prepared and isolated as ahomogeneous substance, nor has there been any report on the physiologalactivity of such a homogeneous substance.

We conducted intensive research on IL-1α as a homogeneous substance andalready established a technique for preparing this substance andclarified the characteristics, physiological activity, etc. thereof.Based on the results of the research, we also confirmed that thepolypeptide of the foregoing formula (A) has LAF activity.

Nevertheless, we have found the surprising fact that the polypeptide,although corresponding to a gene of the living body as reported, isunstable as a substance.

SUMMARY OF THE INVENTION

Accordingly, an object of the present invention is to provide a novelpolypeptide (IL-1α derivative) which differs from the polypeptide of theformula (A) in amino acid sequence, is more stable as a substance andhas suitable characteristics for medicinal uses.

Another object of the present invention is to provide a novel medicinalcomposition comprising the IL-1α derivative.

The present invention provides a gene coding for a novel IL-1αderivative and a process for preparing the IL-1α derivative, i.e.polypeptide, by gene engineering techniques using the gene.

Further the present invention is to provide a novel medicinal use ofIL-1α itself.

More specifically, the present invention provides a polypeptide having amodified amino acid sequence of interleukin-1α represented by theforegoing formula (A) wherein at least one amino acid residue selectedfrom among Ash at the 36 position and Cys at the 141 position isdeficient or replaced by another amino acid.

Amino acids and polypeptide are herein referred to by symbols accordingto the nomenclature or the rules recommended by IUPAC and IUPAC-IUB orby symbols conventionally used in the art. The nucleic acids in basesequences are also similarly expressed.

The IL-1α derivatives of the present invention have, physiologicalactivities such as LAF activity, activity to inhibit growth of tumorcells (GIF activity), i.e. activity to specifically inhibit growth oftumor cells, activity to promote production of various cytokines such ascolony stimulating factor (CSF), interferon (IFN), interleukin-2 (IL-2)and interleukin-3 (IL-3), i.e. activity for example on human cells togreatly promote production of such cytokines, anti-inflammatoryactivity, i.e., activity for example to effectively inhibit progress ofarthritis, when administered to model animals with arthritis, andactivity to prevent radiation injury, i.e. activity to prevent thepossible living body disorders or serious side effects that would resultfrom systemic radiation irradiation during bone marrow transplant,radiation irradiation for treatment of cancers and radiation accident.Accordingly, the present derivatives are very useful as immune systemstimulants, for example, for promoting production of antibodies andenhancing the effect of vaccines, antitumor agents, agents for promotingproduction of cytokines such as CSF, IFN, IL-2 and IL-3,anti-inflammatory agents, agents for preventing radiation injury andother like medicinal agents. Especially, the polypeptide of the presentinvention has the feature of being an exceedingly stable substance andis low in toxicity. Further, IL-1 is known as being pyrogenetic whereasthe polypeptide of the present invention is excellent in being low insuch side effect.

The derivatives of the invention are effective especially as CSFproduction promoting agents. For example, when administered to man, thederivative effectively cures granulocytopenia due to impaired formationof bone marrow resulting from chemotherapy or radiation therapy forcancers without entailing the likelihood of virus infections orantigen-antibody reaction (granulocytopenia curing drug). The CSFproduction promoting agent is usable also for preventing and curingvarious diseases owing to the activity of CSF the production of which ispromoted by the agent as contemplated. For example, CSF acts to promotethe function of granulocytes and macrophages (Lopez, A. F. et al., J.Immunol., 131, 2983 (1983); Handam, E. et al., same, 122, 1134 (1979)and Vadas, M. A. et al., same, 130, 795 (1983)), so that clinicalapplication is expected of CSF for preventing and curing variousinfections. Similarly, the CSF production promoting agent is expected tobe useful for clinical application.

In recent years, it is noted that compromised hosts with impairedbiophylactic ability suffer from so-called opportunistic infections orterminal infections which occur when harmless pathogens becomepathogenic. Clinically, these infections are attributable to pathogensincluding gram-negative bacilli such as Pseudomonas and Serratia,viruses such as Herpes simplex virus (HSV), Varicella-zoster virus (VZV)and Cytomegalovirus (CMV), fungi such as candida albicans, Aspergillusfumigatus and nocardia asteroidea, protozoa such as Pneumocystis cariniiand Toxoplasma gondii, etc. Since the antibiotics presently used are notfully effective on the opportunistic infections, it is desired toconduct research on and develop new drugs for such infections. Thepresent derivatives are effective also for preventing and curingopportunistic infections which occur frequently especially whenanticancer drugs are given. For example, they are useful for preventingand curing various infections which develop during chemotherapy of acuteleukemia and bone marrow transplant, such as candidosis, cryptococcosis,aspergillosis, zygomycosis, chromomycosis, virus infections,Cytomegalovirus pneumonia and complications of these infections.

Further, the polypeptide of the present invention is effective inpreventing and curing arthritis or the like and therefor useful asanti-inflammatory agent.

The polypeptide of the present invention has activity to inhibit growthof tumor cells (hereinafter referred to as "GIF activity") as determinedby the method described in the example to follow, acting specifically onvarious tumor cells to inhibit the growth thereof. Accordingly, theantitumor composition comprising the present polypeptide as its activecomponent is advantageously usable as a chemotherapeutic agent forcancers, especially in combination with various agents for treatingmalignant tumors for a fortified remission therapy and remissionmaintaining therapy.

Moreover, in addition to the foregoing therapeutic purposes, thepolypeptide of the present invention can be effectively used, forexample, in preparing useful cytokines in vitro from cell strain due tothe activity to promote the production of cyctokines. Attention isfocussed on the manufacture of glyccprotein-type cytokines among naturalcytokines produced from cell strain. Useful cytokines can be produced inlarge quantities with efficiency.

Our research discovered novel biological activities of IL-1α. The IL-1αof the invention has GIF activity and activity to promote the productionof CSF, and can be used for said various therapeutic purposes due tothese biological activities.

Among the polypeptides of the invention preferable are those having theamino acid sequence represented by the formula (A) wherein at least Asnat the 36 position is deficient or replaced by another amino acid. Theamino acid capable of replacing Asn at the 36 position and Cys at the141 position can be the residue of any of α-amino acids constitutingliving body proteins. However, in view that Cys is likely to form aninter- or intra-molecular linkage due to the presence of SH groupthereof, preferred amino acids are those other than Cys. Particularly,Asp is preferable for replacing Asn at the 36 position and Ser ispreferable for replacing Cys at the 141 position.

The polypeptides of the present invention can be prepared, for example,by gene engineering techniques using a gene coding for the specificpolypeptide of invention, i.e., by incorporating the gene into amicroorganism vector to effect replication, transcription andtranslation within the cell of the microorganism. This process isadvantageous in that it is amenable to mass production.

Although the gene to be used in this process can be totally synthesizedthrough chemical synthesis of nucleic acids by a usual method, forexample, by the phosphite triester method (Nature, 310, 105 (1984)) orthe like, it is convenient to utilize the gene coding for IL1α or aprecursor thereof. By a conventional method involving the above chemicalsynthesis, the gene is modified to a sequence of nucleic acids codingfor the foregoing specific amino acid sequence, whereby the desired genecan be prepared easily.

The gene coding for IL-1α or a precursor thereof is already known. Weobtained the gene coding for IL-1α and succeeded in preparing IL-1α bygene engineering techniques using this gene. The series of geneengineering techniques employed will be described in the referenceexamples to follow.

The above-mentioned modified sequence of nucleic acids (bases) isprepared also by a known procedure, which executed according to theamino acid sequence of the desired polypeptide (Molecular Cloning, ColdSpring Harbour Laboratory (1982)).

For example, cleavage, ligation, phosphorylation, etc. of DNA can becarried out by usual methods including treatment with enzymes such asrestriction enzymes, DNA ligase, polynucleotidekinase and DNApolymerase, which are readily available as commercial products. Theisolation and purification of the gene and nucleic acids included inthese methods are conducted also in the usual manner, for example, byagarose gel electrophoresis. As will be described partially later, thegene obtained is replicated using a usual vector. The DNA fragmentcoding for the desired amino acid sequence and synthetic linkers can beprepared also easily by the above-mentioned chemical synthesis. Thecodon corresponding to the desired amino acid and to be used in theabove methods is known and is selected as desired. A usual method may beused for this purpose, for example, in view of the frequency of use ofthe codon of the host to be used (Nucl. Acids Res., 9, 43-74 (1981)).Further for the modification of the codon in the nucleic acid sequenceconcerned, for example, site-specific mutagenesis (Proc. Natl. Acad.Sci., 81, 5662-5666 (1984)) can be resorted to as usually done whichemploys a primer comprising a synthetic aligonucieotide coding for thedesired modified sequence about 15-30 mer.

The desired gene obtained by the foregoing process can be checked forits base sequence, for example, by the Maxam-Gilbert chemicalmodification method (Meth. Enzym., 65, 499-560 (1980)) or by thedideoxynucleotide chain termination method using M13 Phage (Messing, J.and Vieira, J., Gene, 19, 269-276 (1982)).

While the above process and procedures therefor will be described in thereference examples and examples to follow, the process is notspecifically limited; any process already known in the art may be used.

Thus, the present invention also provides a novel gene coding for apolypeptide having the above-specified amino acid sequence. (The genewill hereinafter be referred to as the "present gene.")

The polypeptide of the present invention can be prepared by usual knowngene recombination techniques using the present gene. More specifically,it is produced by preparing a recombinant DNA which can express thepresent gene in host cells, transforming the DNA into the host cell andincubating the transformant.

Useful host cells can be either eucaryotic or procaryotic cells. Theeucaryotic cells include cells of vertebrate animals, yeasts, etc.Generally used as cells of vertebrate animals are, for example, COScells which are cells of monkey (Y. Gluzman, Cell, 23, 175-182 (1981)),dihydrofolate acid reductase defective strain of Chinese hamster ovarycell (G. Urlaub and L.A., Chasin, Proc. Natl. Acad. Sci., U.S.A., 77,4216-4220 (1980)), etc., while useful cells are not limited to thesecells. Useful expression vectors of vertebrate cells are those having apromotor positioned upstream of the gene to be expressed, RNA splicingsites, polyadenylation site, transcription termination sequence, etc.These vectors may further have a replication origin when required.Examples of useful expression vectors include pSV2dhfr having an initialpromotor of SV40 (S. Subramani, R. Mulligan and P. Berg, Mol. Cell.Biol., 1(9), 854-864), which is not limitative.

Yeasts are widely used as eucaryotic microorganisms, among which thoseof the genus Saccharomyces are generally usable. Examples of popularexpression vectors of yeasts and like eucaryotic microorganisms includepAM82 having a promotor for acid phosphatase gene (A. Miyanohara et al.,Proc. Natl. Acad. Sci., U.S.A., 80, 1-5 (1983), etc.

E. coli and Bacillus subtilis are generally used as procaryotic hosts.The present invention employs, for example plasmid vectors capable ofreplication in the host. To express the gene in the vector, expressionplasmids can be used which have a promotor and SD (Shine-Dalgarno) basesequence at the upstream of the gene and ATG required for initiatingprotein synthesis. Widely used as host E. coli is E. coli K12 strain.pBR322 is a vector which is generally used. However, these are notlimitative, and various known strains and vectors are usable. Examplesof promotors usable are tryptophan promotor, P_(L) promotor, lacpromotor, lpp promotor, etc. The gene can be expressed with use of anyof these promotors.

To describe the procedure with reference to the case wherein tryptophanpromotor is used, vector pTM1 (Fumio Imamoto, Taisha (Metabolism), Vol.22, 289 (1985)) having tryptophan promotor and SD sequence is used as anexpression vector. A gene coding for a desired polypeptide of thisinvention and having ATG when required is linked to the site ofrestriction enzyme ClaI which is present downstream from the SDsequence.

Incidentally, not only the direct expression system but a fusion proteinexpression system is also usable which employs, for example,β-galactosidase, β-lactamase or the like.

The expression vector thus obtained is introduced into host cells andthereby transformed by usual methods. For example, cells chiefly in thelogarithmic growth phase are collected, treated with CaCl₂ and therebymade to readily accept DNA, whereupon the vector is introducted into thecell. With this method, MgCl₂ or RbCl can be made present conjointlywith the vector so as to achieve an improved transformation efficiency,as is generally known. The cell can be converted to spheroplast orprotoplast before transformation.

The desired transformant thus obtained can be incubated in the usualmanner, whereby the desired polypeptide is produced and accumulated. Themedium for the incubation may be any of those generally used forincubating cells, such as L medium, E medium, M9 medium, etc. Variouscarbon sources, nitrogen sources, inorganic salts, vitamins, etc. whichare usually known can be admixed with these media. When the tryptophanpromotor is used, M9 minimum medium, for example, is usable which hasusually admixed therewith Casamino acid for effecting the action of thepromotor. A chemical, such as indoleacrylic acid, for enhancing theaction of tryptophan promotor can be added to the medium at a suitablestage of incubation.

The desired polypeptide of the present invention can be isolated fromthe resulting culture and purified by usual methods. It is desirable toextract the polypeptide from the host under a mild condition as byosmotic shock in order to maintain the higher order structure thereof.

The above isolation or purification method is conducted substantially bythe same method as usually used for separating a protein-like substancefrom such a biological substance. For example, various procedures areusable utilizing the physical or chemical properties of the desiredpolypeptide. (See for example, "Biological Data Book II," pp. 1175-1259,1st edition, 1st print, Jun. 23, 1980, published by Kabushiki KaishaTokyo Kagakudojin.) Examples of useful procedures are treatment with useof a usual protein precipitating agent, ultrafiltration, molecular sievechromatography (gel filtration), liquid chromatography, centrifugation,e! ectrophoresis, affinity chromatography, dialysis, and combinations ofsuch procedures.

The desired polypeptide is separated from the supernatant as partiallypurified. This partial purification is carried out, for example, by atreatment using as a protein precipitating agent an organic solvent suchas acetone, methanol, ethanol, propanol or dimethylformamide (DMF), oran acidic reagent such as acetic acid, perchloric acid (PCA) ortrichloroacetic acid (TCA), a treatment using a salting-out agent suchas ammonium sulfate, sodium sulfate or sodium phosphate and/orultrafiltration using a dialysis membrane, flat membrane, hollow fibermembrane or the like. These treatments conducuted in the same manner asusually done under usual conditions.

The roughly purified product thus obtained is then subjected to gelfiltration, whereby a fraction exhibiting the activity of the desiredsubstance is collected. Useful gel filtration agents are not limitedspecifically. Such agents include those made of dextran gel,polyacrylamide gel, agarose gel, polyacrylamideagarose gel, cellulose orthe like. Examples of useful agents commercially available are SephadexG type, Sephadex LH type, Sepharose type, Sephacryl type (all productsof Pharmacia Fine Chemicals AB), Cellofine (Chisso Corporation), BiogelP type, Biogel A type (both product of Bio-Rad Laboratories), Ultro gel(LKB Producter AB), TSK-G type (product of Toyo Soda Mfg. Co., Ltd.),etc.

The polypeptide of the present invention can be isolated from thefraction as a homogeneous substance, for example, by subjecting thefraction to affinity chromatography with use of a hydroxyapatite column,ion exchange column chromatography as of the DEAE, CM or SP method,chromatofocusing method, reverse-phase high-performance liquidchromatography or the like, or to a combination of such methods.

The chromatofocusing method can be carried out by various knownprocedures. Usable as the column is, for example, PBE94 (Pharmacia) orthe like, as the starting buffer, for example, imidazole-hydrochloricacid or the like, and the eluent, for example, the mixture of Polybuffer74 (Pharmacia) and hydrochloric acid (pH 4.0) or the like.

The reverse-phase high-performance liquid chromatography can beconducted, for example, with C₄ Hi-Pore reverse-phase HPLC column(Bio-Rad Laboratories) or the like, using acetonitrile, trifluoroaceticacid (TFA), water or the like, or a mixture of such solvents as theeluent.

In this way, the IL-1α derivative (polypeptide) of the present inventioncan be obtained upon isolation. The gene coding for IL-1α affords IL-1αthrough a similar gene recombination procedure.

The IL-1α and derivatives thereof of the invention, which haveoutstanding pharmacological activities as already stated, can beformulated into useful preparations for the afore-mentioned medicinaluses. Examples of such medicinal preparations include immunostimulatorsfor producing antibodies, enhancing the effect of vaccines and curingimmunodeficiency, antitumor agents, cytokine production promotors,anti-inflammatory agents, agents for preventing or curing radiationsickness, agents for preventing or curing opportunistic infections, etc.These medicinal preparations are formulated usually in the form ofpharmaceutical compositions comprising a pharmacologically effectiveamount of the IL-1α or derivative thereof of the present invention and asuitable carrier. Examples of useful pharmaceutical carriers includeexcipients and diluents such as filler, extender, binder, wetting agent,disintegrator, surfactant, etc. which are generally used for preparingpharmaceuticals of the desired form to be used. The form of thepharmaceutical compositions is not specifically limited insofar as theyeffectively contain the present polypeptide or IL-1α but can be, forexample, in the form of tablets, power, granules, pellets or like solidpreparation. Usually, however, it is suitable that the composition be inthe form of a solution, suspension, emulsion or the like for injection.Alternatively, such a composition can be a dry product which can be madeliquid with addition of a suitable carrier before use. Thepharmaceutical compositions mentioned above can be prepared by usualmethods.

In accordance with the form of the pharmaceutical composition obtained,the composition is administered via a suitable route. For example, thosefor injection are given intravenously, intramuscularly, subcutaneously,intracuteneously, intraperitoneally or otherwise. The solid compositionis given orally or intraintestinally. The amount of the active componentof the composition and the dosage of the composition are suitablydetermined according to the method and form of administration, purposeof use, symptoms of the patient, etc. and are not definitelydeterminable. It is generally desirable to incorporate about 1 to about80 wt. % of the active component into the pharmaceutical preparation andto give the preparation at a daily dose of about 0.1 μg to about 10 mg,calculated as the active component, for adults. The preparation need notalways be given only once a day but can be given in three to fourdivided doses daily.

The present invention will be described in greater detail with referenceto the following examples.

In the following examples, physiological activities were determined bythe following methods.

(1) Determination of IL-1 Activity

Expressed in terms of LAF activity as measured by the method of J. J.Oppenhein et al. (J. Immunol., 116, 1466 (1976)) using thymus cells of amouse of C3H/HeJ strain.

(2) Determination of GIF Activity

Portions (0.1 ml) of the test solution diluted to varying concentrationswere placed into the wells of 96-well microplate (Corning Glass Works),0.1 ml of Eagle's MEM suspension containing 10% FCS containing humanmelonoma cells A375 in an amount of 2 ×10⁴ cells/ml was then placed intoeach well, and the cells were incubated in a CO₂ incubator (Napco Co.,Ltd.) for 4 days. After the incubation, 0.05 ml of 0.05% Neutral Red(Wako Pure Chemical Ind. Ltd.) was placed into each well, followed byincubation at 37° C. for 2 hours. After removing the supernatant, 0.3 mlof phosphoric acid buffer saline was gently poured into each well forwashing. After removing the washing, 0.1 ml of mixture of sodiumphosphate monobasic and ethanol in equal amounts was placed into eachwell, the plate was shaken for several minutes by a 10 micromixer, andthe amount of pigment taken into the cell was measured at an absorbanceof 540 mμ using a photometer for 96-well microtitration plates (Titercheck multiscane, Flow Lab.) to determine growth inhibition activity.The test group exhibiting 50% of the inhibition of cell growth of thecontrol group, i.e., the test group which exhibited 1/2 the absorbancemeasured of the control group, was identified. The reciprocal of thenumber of times of dilution for the test group was taken as the GIFactivity unit. Accordingly, when the GIF activity is 10 units, forexample, the test solution, if diluted tenfold, still has activity toinhibit cell growth 50%.

The following drawings are referred to in reference examples andexamples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows restriction enzyme maps of cDNA of plasmid pcD-GIF-16 andplasmid pcD-GIF-207;

FIG. 2 is a diagram showing how plasmid ptrpIL-1α-113 is constructedfrom plasmid pcD-GIF-207 and plasmid pTM1;

FIG. 3 shows the results of an antitumor activity test;

FIGS. 4 and 5 show the results obtained by testing the polypeptide ofthe invention for effect to promote production of CSF;

FIG. 6 shows the results obtained by testing the polypeptide of theinvention for anti-inflammatory effect;

FIG. 7 shows the results obtained by testing the polypeptide of theinvention for activity to prevent radiation injury; and

FIG. 8 shows the results obtained by testing the polypeptide of theinvention for activity to prevent opportunistic infection.

REFERENCE EXAMPLE 1 (1) Incubation of U937 Cells

Human histiocytic lymphoma U937 cells (Ascenso, J. L. et al., Blood,Vol. 57, p170 (1981)), 1.4×1O⁹ in number, were placed into RPMI-1640culture medium containing 25 ng/ml of12-o-tetradecanoylphorbol-13-acetate (TPA, product of Pharmacia), 10μg/ml of concanavalin A (ConA, product of Sigma Chemical. Co.) and 10%FCS to prepare a cell suspension having a concentration of 4×10⁵cells/ml.

Ten-ml portions of the cell suspension were separately placed intodishes (Falcon 3003), 9 cm in diameter, and incubated in 5% carbondioxide gas at 37° C. for 3 days. After removing the culture supernatantby an aspirator, 10 ml of RPMI-1640 medium containing 10% FCS, 10 μg/mlof bacterial lipopolysaccharide (LPS, product of Difco), 1 μg/ml ofmuramyldipeptide (MDP, product of Wako Pure Chemical Ind. Ltd.) and 1ng/ml of TPA was placed into each dish. The cells were incubated in thismedium in 5% carbon dioxide gas at 37° C. for 18 hours. The U937 cellsadhering to the bottom of the dish were used for preparing mRNA.

(2) Preparation of mRNA

RNA was extracted by the combination of the guanidinium/hot phenolmethod (Feramisco, J. R. et al., J. Bio. Chem., Vol. 257, 11024 (1982))and the guanidinium/cesium chloride method (Glisin, V, et al.,Biochemistry, Vol. 13, 2633 (1974)).

To wash the U937 cells incubated by the procedure (1), the dishes wererinsed with 5 ml of PBS(-) solution after removing the supernatant. Thecells were then dissolved with 1 ml of 4M guanidine isothiocyanatesolution (4M guanidine isothiocyanate (product of Fluka AG.), 50 mMtris-HCl (pH 7.6), 10 mM EDTA and 2% sodium lauroyl sarkosinate placedinto each dish. The solution was collected with rubber polisher andPasteur pipette to obtain 420 ml of cell solution, which was maintainedat 60° C. and passed through 18 G injection needle to shear thechromosome DNA. Subsequently, phenol heated to 60° C. was added to thesolution in an amount equal to that of the solution, and the mixture wasstirred with 18 G injection needle for further shearing. To the mixturewere then added 210 ml of 0.1M sodium acetate-10 mM tris-HCl (pH 7.4)-1mM EDTA solution and 420 ml of chloroformisoamyl alcohol mixture (24:1in volume ratio). The resulting mixture was vigorously agitated at 60°C. for 15 minutes, then ice-cooled and centrifuged at 3000 r.p.m. at 4°C. for 20 minutes. To the aqueous layer collected was added ethanol intwo times the amount of the aqueous layer. The mixture was allowed tostand overnight at -70° C. to obtain crude RNA precipitate. The crudeRNA was dissolved in 48 ml of 6M guanidine isothiocyanate-5 mM sodiumcitrate (pH 7.0)-0.1 M β-mereaptoethanol-0.5% sodium lauroyl sarkosinatesolution. Cesium chloride (19.2 g) was then dissolved in the solution.Seven-ml portions of the resulting solution was then superposed on 4 mlof 5.7M cesium chloride-0.1M EDTA (pH 7.5). The mixture was centrifugedat 31500 r.p.m. at 25° C. for 20 hours by Beckman SW40Ti rotor tocollect RNA.

Thus, 9.7 mg of RNA was obtained.

To obtain mRNA from the RNA, the RNA was subjected to columnchromatography using oligo(dT)-cellulose (product of CollaborativeResearch Inc.). For adsorption, 10 mM tris-HCl (pH 7.5)-0.5M NaCl-1 mMEDTA was used. For elution, 10 mM tris-HCl (pH 7.5)-1 mM EDTA was used.

Consequently, 400 μg of mRNA was obtained.

(3) Preparation of cDNA Library

cDNA library was prepared by the Okayama-Berg method by which cDNA canbe expressed in animal cells. Thus, dT tail-attached vector primer foruse in cDNA cloning was prepared from plasmid pcDVl, and dGtail-attached linker DNA from plasmid pLl, each by the method of Okayamaet al. (Okayama, H. and P. Berg, Molecular and Cellular Biology, Vol. 3,p. 280 (1983)).

mRNA (15 μg) obtained by the procedure (2) was dissolved in 20 μl of 5mM tris-HCl (pH 7.5)-0.5 mM EDTA (pH 7.5) aqueous solution and incubatedat 65° C. for 5 minutes and then at 37° C. for 5 minutes. The reactionmixture was adjusted to a total quantity of 40 μl containing 50 mMtris-HCl (pH 8.3), 8 mM MgCl₂, 30 mM KC1, 0.3 mM dithiothreitol, 2 mM ofeach of dATP, dGTP, dCTP and dTTP, 2.8 μg of vector primer DNA, 60 unitsof RNase inhibitor (product of Promega Biotech) and 40 units of reversetransferase (product of Bio-Rad), followed by incubation at 37° C. for 1hour. The reaction was terminated by addition of 2 μl of 0.5M EDTA (pH7.5) and 2 μl of 10% SDS. Subsequently, the mixture was subjected tophenol-chloroform extraction and chloroform extraction, and the extractwas precipitated from ethanol to collect vector primer cDNA:mRNA.

The collected vector primer cDNA:mRNA was incubated at 37° C. for 5minutes in 30 μl of a reaction mixture composed of 140 mM sodiumcacodylate, 30 mM tris-HCl (pH 6.8), 1 mM CaCl₂, 0.1 mM dithiothreitol,0.3 μg of poly A, 66 μM dCTP and 38 units of terminal deoxynucleotidyltransferase (product of Pharmacia), whereupon 1.5 μl of 0.5M EDTA (pH7.5) and 1.5 μl of 10% SDS were added to the mixture to terminate thereaction. The mixture was subjected to phenol-chloroform extraction andchloroform extraction, and the extract was precipitated from ethanol tocollect oligo dC tail-attached cDNA:mRNA-vector primer.

The collected nucleic acid was incubated at 37° C. for 90 minutes in 20μl of a reaction mixture comprising 7 mM tris-HCl (pH 7.5), 7 mM MgCl₂,60 mM NaCl, 100 μg/ml of bovine serum albumin and 12 units ofrestriction enzyme HindIII (product of Nippon Gene Co., Ltd.)Subsequently, 1 μl of 0.5M EDTA (pH 7.5) and 1 μl of 10% SDS were addedto the mixture to terminate the reaction, followed by extraction withphenol-chloroform and then with chloroform and by precipitation fromethanol to collect HindIII-digested oligo dC tail-attachedcDNA:mRNA-vector primer. The product was dissolved in 10 μl of 10 mMtris-HCl (pH 7.5)-1 mM EDTA (pH 7.5) (TE (pH 7.5)). One μl portion ofthe solution was incubated in 10 μl of a reaction mixture comprising theabove TE (pH 7.5), 0.1M NaCl and oligo dG tail-attached linker DNA (14ng), first at 65° C. for 2 minutes and then at 42° C. for 30 minutes.The mixture was thereafter cooled to 0° C.

The reaction mixture was adjusted to 100 μl containing 20 mM tris-HCl(pH 7.5), 4 mM MgCl₂, 10 mM (NH₄)₂ SO₄, 0.1M KCl, 50 μg/ml bovine serumalbumin, 0.1 mM β-NAD (nicotinamide-adenine-dinucleotide, product ofPharmacia) and 0.6 μg of E. coli DNA ligase (product of Pharmacia) andincubated at 12° C. overnight. To the reaction mixture were added 40 μMof each of dATP, dGTP, dCTP and dTTP and 0.15 mM of β-NAD. Further withaddition of 0.4 μg of E. coli DNA ligase, 4.6 units of E. coli DNApolymerase I (product of Boehringen Mannhein) and 1 unit of E. coliRNase H (product of Pharmacia), the mixture was incubated at 12° C. for1 hour and then at 25° C. for 1 hour.

E. coli HB101 was transformed using the reaction mixture thus obtained.The competent cells of this strain used were a product of BethesdaResearch Laboratories (BRL). The cells were transformed according toBRL's manual.

Consequently, cDNA library was obtained which contained about 21000clones.

(4) Transfection of Monkey COS-1 Cells

The cDNA library obtained by the above procedure (3) was divided intogroups each including about 70 clones on the average. Plasmid DNA wasprepared from each group.

The plasmid DNA was prepared by the alkaline lysis method (MolecularCloning-A Laboratory Manual, Cold Spring Harbor Laboratory, 1982, p.368).

Incubated monkey cells, COS-1 cells (Gluzman, Y., Cell, Vol. 23, p. 175(1981)), were infected with the plasmid DNA thus prepared from eachgroup for transfection. The transfection was conducted by theDEAEdextran method (Yokota, T. et al., Proc. Natl. Acad. Sci. U.S.A.,Vol. 81, p. 1070 (1984)). More specifically, the COS-1 cells treatedwith trypsin were suspended in RPMI-1640 medium containing 10% FCS andadjusted to a concentration of 1×10⁶ cells/ml. Subsequently, 500 μlportions of the suspension were placed into 6 wells of a well plate eachcontaining 2 ml of RPMI-1640 medium containing 10% FCS. After incubatingthe cells at 37° C. overnight, the supernatant was removed, the cellswere washed with a medium free from serum, and 1 ml of RPMI-1640 mediumcontaining 10 μg/ml of plasmid DNA, 0.4 mg/ml of DEAE-dextran (productof Pharmacia), 50 mM tris-HCl (pH 7.4) and 10% FCS was placed into eachwell, follwed by incubation at 37° C. for 4.5 hours. The supernatant wasthereafter removed, the cells were washed with a medium free from serumand 2 ml of RPMI-1640 medium containing 150 μM chloroquine (product ofSigma) and 10% FCS was placed into each well, further followed byincubation at 37° C. for 3 hours. The supernatant was removed, and thecells were washed with a medium free from serum and thereafter incubatedat 37° C. for 72 hours with addition of 3 ml of RPMI-1640 mediumcontaining 10% FCS to the cells in each well. After collecting thesupernatant, 2 ml of RPMI-1640 medium containing 10% FCS was placed intoeach well, followed by two freeze-thaw cycles. The cell extract was thencollected. The culture supernatant and the cell extract were checked forGIF activity.

The group exhibiting GIF activity was divided into 24 groups of 10clones each, and the same procedure as above was repeated using thesegroups for the determination of GIF activity. The same procedure asabove was repeated for the clones within the group exhibiting GIFactivity to identify the clone exhibiting GIF activity.

Consequently, two kinds of clones, i.e. pcD-GIF-16 and pcD-GIF-207, wereobtained.

Table 1 shows the GIF activity (GIF units/ml) of these clones.

                  TABLE 1                                                         ______________________________________                                        Clone         Culture supernatant                                                                         Cell extract                                      ______________________________________                                        pcD-GIF-16    48.3          120.7                                             pcD-GIF-207   62.6          196.9                                             pcDV1 (control)                                                                             0              0                                                ______________________________________                                    

(5) Analysis of Clone

FIG. 1 shows restriction enzyme maps of the cDNA of plasmid pcD-GIF-16and plasmid pcD-GIF-207.

The base sequence of the cDNA of each of these plasmids was determinedby the base-specific chemical modification method (Methods inEnzymology, Vol. 65, p. 499 (1980)) and dideoxy chain termination (Proc.Natl. Acad. Sci., U.S.A., Vol. 74, p. 5463 (1977)) using phage M13vector [Gene, Vol. 19, p. 269 (1982)].

Consequently, cDNA of pcD-GIF-207 was found identical with cDNA in thecoding region of IL-1α reported by March et al. (Nature, Vol. 315, p.641 (1985)).

E. coli strain x1776 harboring plasmid pcD-GIF-207 having cDNA codingfor a precursor protein of IL-1α has been deposited under the name of"Escherichia coli .sub.χ 1776/pcD-GIF-207" with deposition number FERMBP1294 in Fermentation Research Institute, Agency of Industrial Science& Technology.

(6) Expression and Preparation of Polypeptide

The above plasmid, pcD-GIF-207, was cleaved with restrcition enzymesHindIII and HincII and then electrophoresed on agarose gel to isolateand purify about 0.66-kb HindIII-HincII DNA fragment, which was furthercleaved with restriction enzyme AluI to similarly obtain about 0.5-kbAluI-HincII DNA fragment on isolation.

Next, synthetic oligonucleotides (5' CGATAATGTCAGCACCTTTTAG 3' and 5'CTAAAAGGTGCTGACATTAT 3') were each phosphorylated at the 5' terminalwith T4 polynucleotide kinase and ligated with AluI-HincII DNA fragmentobtained above, using T4 DNA ligase. The ligated block was then cleavedwith restriction enzyme ClaI and electrophoresed on agarose gel toisolate and purify about 0.54-kb ClaI-ClaI DNA fragment.

On the other hand, plasmid pTMI (Fumio Imamoto, Taisha (Metabolism),Vol. 22, 289 (1985)) was cleaved with restriction enzyme ClaI, reactedwith calf intestine alkaline phosphatase (CIAP) and thereafter ligatedwith the approximately 0.54-kb ClaI-ClaI DNA fragment previouslyprepared, using T4 DNA ligase to obtain the desired polypeptideexpression plasmid ptrpIL-1β-113.

This plasmid was transformed into E. coli HB101, and the desiredtransformant was selected by analyzing by the boiling method the size ofcut-off fragments obtained from the resulting plasmid DNA.

FIG. 2 schematically shows the above procedure.

Plasmid ptrpIL-1α-113 was extracted from the selected transformant andtransformed into E. coli W3110 to obtain E. coli W3110/ptrpIL-1α-113.

EXAMPLE 1 (1) Incubation of Transformant

The transformant (E. coli W3110/p trpIL-1α-113) obtained in ReferenceExample 1, (6) was incubated with shaking at 37° C. overnight in 10 mlof LB medium (1% tryptone, 0.5% yeast extract and 0.5% NaCl) containing50 μg/ml of ampicillin and 20 μg/ml of L-tryptophan. A 400 ml quantityof M9 minimum medium (0.6% Na₂ HPO₄, 0.3% KH₂ PO₄, 0.05% NaCl, 0.1% NH₄Cl, 2 mM MgSO₄, 0.2% glucose and 0.1 mM CaCl₂) containing 50 μg/ml ofampiciilin and 1% Casamino acid was inoculated with 8 ml of the culture,followed by incubation at 37° C. for 9 hours. The E. coli cells obtainedwere suspended in 10 ml of 1M Na₂ HPO₄ and allowed to stand overnight ina cold chamber. The suspension was then dialyzed against 10 mM tris-HClbuffer (pH 8.0) for 2 days.

The dialyzate obtained was centrifuged and thereby separated into asupernatant and a sediment. The supernatant obtained in an amount of 28ml was found to have GIF activity of 7.3×10⁷ units.

(2) Purification of Polypeptide

A 2 ml quantity of the supernatant obtained by the above procedure (1)was purified under the following conditions by ion-exchange highperformance liquid chromatography (DEAE-HPLC) using ULTROCHROM GTi (LKB)chromatography system.

Column: TSK gel DEAE-5PW (7.5×75 mm, product of Toyo Soda Mfg. Co.,Ltd.)

Eluent A: 20 mM tris-HCl buffer (pH 8.0)

Eluent B: 20 mM tris-HCl buffer (pH 8.0) containing 0.5M NaCl

Flow rate: 1 ml/min

Fraction volume: 1 ml/min/tube

    ______________________________________                                        Gradient profile:                                                                    Time (min)                                                                            % B                                                            ______________________________________                                                0      0                                                                      5      0                                                                     65      60                                                                    70      100                                                                   80      100                                                                   85      0                                                                     100     0                                                              ______________________________________                                    

The DEAE-HPLC procedure afforded two GIF-active fractions, i.e. afraction of 26 to 28 min in retention time (hereinafter referred to as"fraction 1") and another fraction of 32.5 to 34.5 min in retention time(hereinafter referred to as "fraction 2").

These two fractions were subjected to DEAE-HPLC again and to gelfiltration-HPLC to obtain fractions 1 and 2 as purified.

Since the divided GIF-active fractions were obtained as above, thesefractions were tested as follows for confirmation.

(3) SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Fractions 1 or 2 were subjected to SDS-PAGE by the method of U.K.Laemmli (Nature, 277, 680 (1970)) under the following conditions.

Specimen: Fraction 1 or 2 was completely dried, then dissolved inLaemmli sample buffer (containing 2-mercapto ethanol (2ME⁺) in an amountof 1/20 the volume of the buffer) and treated at 100° C. for 4 minutes.

Gel: 15% polyacrylamide gel, 1.5 mm in thickness.

Apparatus: PROTEAN, product of Bio-Rad.

Electrophoresis: 40 mA constant current for 2 hours.

The gel resulting from the electrophoresis was stained with Silver StainKit (Bio-Rad). Consequently, each of fractions 1 and 2 migrated as asingle band at a molecular weight of about 18.3 kd, which nearly matchedthe molecular weight of 18 kd calculated from the gene.

(4) Isoelectrofocusing (IEF)

Fractions 1 and 2 were subjected to IEF using ampholine PAG plate (LKB),3.5 to 9.5 in pH range, and Model 1415 (Bio-Rad) under the followingconditions. Specimens:PBS solution of fraction 1 as allowed to stand for2 days, the same as allowed to stand for 2 weeks, PBS solution offraction 2 as allowed to stand for 2 weeks, PBS solution serving as acontrol, and the following marker proteins (pI marker proteins), fivelanes in total.

Marker proteins

Amyloglucosidase (3.50)

Soybean trypsin inhibitor (4.55)

β-lactoglobulin A (5.20)

Bovine carbonic anhydrase B (5.85)

Human carbonic anhydrase B (6.55)

Horse myoglobin-acidic band (6.85)

Horse myoglobin-basic band (7.35)

Lentil lectin-acidic band (8.15)

Lentil lectin-middle band (8.45)

Lentil lectin-basic band (8.65)

Trypsinogen (9.30)

Electrode solutions:

Anode solution=1M H ₃ PO₄

Cathode solution=1M NaoH Electrophoresis: With constant power of 1 W/cmgel width with cooling (10° C.) for 90 minutes.

Staining: With Silver Stain Kit

The gel resulting from the electrophoresis was sliced at a spacing of 1cm and subjected to extraction with 1 ml of distilled water with shaking(2 days). The isoelectric point was calculated from the pH measurementafter the electrophoresis.

The isoelectric point (pI) of fraction 2 was found to be about 5.0, andthe fraction migrated as a single band at this position. Fraction 1 hadisoelectric points of about 5.2 and about 5.0 and migrated as two bands.

(5) Amino Acid Composition

Each of fractions 1 and 2 (30 μl) was carefully placed into the bottomof a thick-walled hard test tube made of Pyrex glass and 12 mm×120 mm,and was dried in a vacuum in a desiccator containing sodium hydroxidepellets. A 50 μl quantity of 4N methane-sulfonic acid (containing 0.2%3-(2-aminoethyl)indole and produced by Pierce) was added to the dryspecimen within the tube. The tubes were deaerated at 0.1 to 0.2 mm Hgfor 1 minute and then sealed off. The specimen was hydrolyzed in aheater at 118° C. over a period of 24 hours. After opening the tube, themixture was neutralized with 46 μl of 4N sodium hydroxide and diluted toan amount of 450 μl with citric acid buffer.

A 250 μl quantity of the specimen solution was used for amino acidanalysis by an amino acid analyzer (Model 835, product of Hitachi Ltd.).The amino acids separated were detecteds by the o-phthalaldehyde methodand quantitatively determined with reference to calibration curvesprepared with use of authentic amino acids.

Table 2 shows the results in terms of the mole ratio of component aminoacids based on Phe (10 moles). Under the above analysis conditions, Proand Cys are not determinable.

                  TABLE 2                                                         ______________________________________                                        Amino acid    Fraction 2 (mole ratio)                                         ______________________________________                                        Asp and/or Asn                                                                              20.6                                                            Thr           11.4                                                            Ser           10.1                                                            Glu and/or Gln                                                                              16.8                                                            Gly           6.6                                                             Ala           14.4                                                            Val           5.8                                                             Met           2.3                                                             Ile           9.3                                                             Leu           15.0                                                            Tyr           6.8                                                             Phe           (10)                                                            Lys           10.5                                                            His           3.0                                                             Trp           1.6                                                             Arg           2.9                                                             ______________________________________                                    

(6) Amino Acid Sequence

Each of fractions 1 and 2 (150 μl) was analyzed by a protein sequencer(Model 470A, Applied Biosystems Inc.). Each resulting PTH-amino acid wassuitably diluted with 100 to 50 μl of 33% aqueous acetonitrile solution,and a 5-μl portion of the dilution was injected into a chromatographiccolumn by an autosampler, Waters 710B. For the chromatographic system,two pumps, Beckman Model 112, were operated by a controller, Model 421.The column used, measuring 2 mm×250 mm and packed with Ultrasphere ODS-5μm, was maintained at 55° C. by a column heater. The flow rate was 0.3ml/min. A mixture of 20 mM sodium acetate and acetonitrile was used forgradient elution. Absorbance was monitored at 269 nm. Analysis wasconducted for 45 minutes.

The results of 40 cycles of analysis revealed that the two fractionswere identical in amino acid sequence except the 36th amino acid andthat they had the following sequence. ##STR2##

The 36th amino acid (X in the above formula) of fraction 1 was Asn whichhad been predicted from the sequence of the gene, and that of fraction 2was Asp.

The foregoing indicates that fraction 1 is the polypeptide of theformula (A), namely IL-1α, further revealing that the other fraction 2is a polypeptide of the invention which corresponds to IL-1α wherein the36th amino acid is replaced by Asp. This polypeptide of the inventionwill be hereinafter referred to as "polypeptide I."

The IL-1α was unstable as a substance and was found, for example, topartly change into a more stable polypeptide I when present in a trisHClbuffer (pH 8.0).

EXAMPLE 2 (1) Preparation of Polypeptide I of the Invention

A polypetide I expression plasmid was prepared by the site-specificmutagenesis method (Pro. Nat. Acad. Sci., 81, 5662-5666 (1984); Science,224, 1431 (1984)), using plasmid ptrpIL-1α-113 obtained in ReferenceExample 1, and polypeptide I was prepared from the plasmid as will bedescribed below.

M13 mp 11 phage vector was used as a single-strand DNA template.EcoRI/BamHI DNA fragment was isolated from plasmid ptrpIL-1α-113 andcloned in M13 mp 11 phage (RF) at restriction enzyme EcoRI and BamHIsites to obtain single-strand (ss) DNA (M13-IL-1α-113), which was thenused as a mutagenesis template.

Synthetic oligonucleotide [5'-ACTGGGTGAGCTTGGCAG-3' (primer)] wasphosphorylated with T4 polynucleotide kinase and hybridized with ssM13-IL-1α-113 DNA. The hybrid was annealed, thereafter treated with DNApolymerase I, (Klenow fragment) and T4 DNA ligase in the presence ofdNTPs and incubated at 15° C. for 18 hours.

The DNA obtained was introduced into JM105 competent cell fortransformation. The resulting phage plaque (50 colonies) was inoculatedonto the intro-cellulose filter on the agar plate and incubated at 37°C. for 18 hours. A filter containing the cloning was treated with analkali in the usual manner for denaturation, dried and then baked at 80°C. for 2 hours. The filter was prehybridized and then hybridized at roomtemperature with ³² P probe prepared by labeling the primer with ³²P-γ-ATP. The filter hybridized was washed with 6× SSC buffer at roomtemperature for 10 minutes and further at 54° C. for 5 minutes, driedand thereafter subjected to autoradiography at -70° C. for 18 hours.

M13-IL-1α-36D was selected as a typical clone from among five mutantclones, infected with JM105 and incubated to prepare ssDNA and RF DNA.

M13 dideoxynucleotide sequencing was performed for ssDNA to confirmmutation of the contemplated gene.

EcoRI/BamHI fragment was prepared from RF DNA produced in JM105 andintroduced into expression plasmid in the same manner as in theforegoing reference example to obtain the desired polypeptide Iexpression plasmid (IL-1α-36D).

Using this plasmid, polypeptide I was expressed in the same manner as inExample 1, followed by purification (DEAE-HPLC gave a single GIFactivity peak), whereby the desired polypeptide I was obtained with theforegoing characreristics. The product was found to have specificactivity of about 1×10⁷ GIF units/mg protein.

EXAMPLE 3 (1) Preparation of Polypeptides II and III of the Invention

Plasmid ptrpIL-1α-141S for expressing a polypeptide of the invention,corresponding to IL-1α wherein the 141st amino acid (Cys) is replaced bySer, was prepared in the same manner as in Example 2 using5'-ACTGGGTGAGCTTGGCAG-3' as the primer.

The same expression and purification procedures as in Example 1 wererepeated using this plasmid, DEAE-HPLC similarly conducted gave two GIFactivity peaks. The peak fractions were analyzed in the same manner asabove, with the result that the peak fraction which was earlier inretention time was identified as a polypeptide of the invention(polypeptide III) anticipated from the gene sequence, i.e. onecorresponding to IL-1α wherein the 141st amino acid (Cys) was replacedby Ser.

The analysis revealed that the other peak fraction was a polypeptide ofthe invention corresponding to IL-1α wherein the 36th amino acid (Asn)and the 141st amino acid (Cys) were replaced by Asp and Ser,respectively. This polypeptide will hereinafter be referred to as"polypeptide II."

EXAMPLE 4 (I) Preparation of Polypeptide II of the Invention

Plasmid ptrpIL-1α-36D·141S for expressing the desired polypeptide II wasprepared in the same manner as in Example 3, using plasmidptrpIL-1α-141S obtained in Example 3 and using 5'-ATTCGAGCCGATGATCAG-3'as the primer.

HB101 strain was caused to harbor the above plasmid, ptrpIL-1α-36D·141S.The strain has been deposited under the name of "Escherichia coliHB101/IL-1α-36D141S" with deposition number FERM BP-1295 in FermentationResearch Institute, Agency of Industrial Science & Technology.

The same expression and purification procedures as in Example 1 wererepeated with use of the above plasmid to obtain the desired polypeptideII of the invention.

SDS-PAGE indicated that the polypeptide was about 18 kd in molecularweight. The isoelectric point (PI) thereof was found to be about 5.0 byIEF.

Polypeptides II and III were equivalent to polypeptide I in specificactivity (GIF activity).

PHARMACEUTICAL TEST EXAMPLE 1 (1) GIF Activity and LAF Activity

The GIF activity of the present polypeptide has already been described.The LAF activity of the polypeptides I, II and III was comparable to theGIF activity thereof.

The polypeptide of the invention was tested as follows.

(2) Test for Antitumor Activity

Tumor cells (200,000), "Meth A", were intracutaneously transplanted intoeach of BALB/c mice (zero day). On the 7th day and 8th day aftertransplantation, the polypeptide I was given to the tumor site of themice in the first experiment in an amount of 0.3, 1 or 3 μg per mouse.

Seven mice in each group were used. A solvent (1% mouse serum) wasadministered in the same manner to the control group.

The test results are shown in FIG. 3, in which the number of dayselasped after the inoculation of tumor cells is plotted as abscissa vs.the weight of the tumor (mg) as ordinate. A control group is representedby curve (1), a group with a dose of 0.3 μg of polypeptide I by curve(2), a group with a dose of 1 μg of polypeptide I by curve (3), and agroup with a dose of 3 μg of polypeptide I by curve (4). The weight oftumor is expressed in terms of mean ± standard deviation (SD). Themark * represents P<0.05 in Students' T-test, and the mark ** P<0.01.

(3) Test for Effect to Promote CSF Production

(3) -1 The following test was conducted using cell strain U-373MG (ATCCHTB17, glioblastoma, astrocytoma, human).

Cells of the above strain were suspended in Eagle's MEM medium (productof Nissui Pharm. Co.) containing 10% FCS (product of GIBCO), MEMnon-essential amino acids (product of Flow) and MEM sodium pyruvate(product of Flow), to a concentration of 2×10⁵ cells/ml. The polypeptideI to be tested was added in varying concentrations to portions of thesuspension. Each of the mixtures was incubated in a carbon dioxideincubator at 37° C. for 24 hours.

The culture supernatant was collected, and the amount of CSF producedand accumulated in the supernatant was measured using mouse bone marrowcells (Lewis, I. C. et al., J. Immunol, 128, 168 (1982)).

The results are given in FIG. 4, in which the concentration (ng/ml) ofpolypeptide I is plotted as abscissa vs. the CSF activity (U/ml,×10⁻²)as ordinate.

(3)-2 The following animal experiment was conducted to substantiate thatpolypeptide I, when administered to the living body, acts to promoteproduction of CSF in vivo.

To normal mice (BALB/C strain, purchased from Experimental AnimalCooperative Association of Shizuoka Prefecture, Japan) was intravenouslygiven polypeptide I in varying amounts. Blood was collected from theanimal 8 hours after the administration and checked for the CSF activityas in (3)-1 above. As a control, human serum albumin (HSA) wasadministered in the same manner to a control group for the test.

The results are shown in FIG. 5 in which the amount of the polypeptide Ior the amount of HSA administered (μg/mouse) is plotted as abscissa andCSF activity (unit/ml serum,×10⁻³) as ordinate.

(4) Test for Anti-inflammatory Effect

The following test was conducted according to the method of Winter etal. (Proc. Soc. Exptl. Biol. Med., 111, 544-547 (1962)).

Six- to eight-week-old male rats (Spraque Dawley strain, Nippon CharlesRiver Co., Ltd.) were used as divided into groups of 6 to 8 rats eachaccording to the body weight one day before the experiment. A suspensionof 1% Carrageenan (product of Marine Colloid) in saline, serving as anagent for causing inflammation, was subcutaneously injected in an amountof 0.1 ml into the sole of the right rear leg of the rat to causeswelling of the foot. To evaluate the swelling, the volume of the solewas measured a predetermined period before and after the injection usinga plethysmometer (product of Ugo-Vasile). The ratio of the increasedvolume due to the injection relative to the value before the injectionwas calculated as swelling %.

The substance to be tested was dissolved in Dulbeco's phosphate bufferedsaline, and 0.1 ml of the solution was subcutaneously injected into theback of the rat 1 hour before the injection of the inflammation causingagent. The solvent was given to a control group.

The results are shown in FIG. 6, in which the time (hours) elapsed afterthe injection of the inflammation causing agent is plotted as abscissavs. the swelling % as ordinate. In the diagram, the control group isrepresented by curve (1), a group to which 0.1 μg of polypeptide I wasgiven by curve (2), a group with 1 μg of polypeptide I by curve (3), anda group with 10 μg of polypeptide I by curve (4).

(5) Test for Effect to Prevent Radiation Injury

One μg or 0.3 μg of polypeptide I was intraperitoneally given to each of9-week-old mice of BALB/c strain 20 hours before the mice were exposedto a lethal dosage of X-rays.

The mice were systemically exposed to X-rays at a dose of 850 roentgensby an X-ray irradiator (MBR-1505R, product of Hitachi Medico Co., Ltd.)and thereafter checked for survival daily. PBS was given to a controlgroup.

The results are shown in FIG. 7, in which the number of days after theirradiation is plotted as abscissa vs. the survival ratio (%) asordinate. The group to which 1 μg of polypeptide I was given isrepresented by curve (1), the group with 0.3 μg of polypeptide I bycurve (2), and the control group by curve (3).

FIG. 7 shows that all the mice of the control group died on the 18th dayafter the X-ray irradiation, whereas polypeptide I was found effectivefor preventing radiation injury depending on the dosage. It was foundthat about 80% of the group with the dose of 1 μg were saved from deathdue to radiation injury and survived.

(6) Test for Effect to Prevent Opportunistic Infection

The following test was conducted using model mouse.

On the first day, 100 mg/kg of 5-fluorouracil (5-Fu, product of KyowaHakko Co., Ltd.) was intravenously given to 6-week-old male mice of ICRstrain (7 mice in each group). On the 2nd, 4th and 6th days, polypeptideI of the invention was subcuteneously administered to the mice at a doseof 1 μg/mouse. On the 7th day, a specified quantity of Pseudomonasaeruginosa E-2 was intraperitoneally given to the mice for infection. Onthe 10th day, the number of animals survived was counted to determinethe survival ratio (%).

The results are given in FIG. 8, (1) to (3) FIG. 8 (1) shows the resultachieved by the group thus treated. FIG. 8 (2) shows the result achievedby a control group to which polypeptide I was not given (but 5-Fu onlywas given). FIG. 8 (3) shows the result achieved by another controlgroup to which neither 5-Fu nor polypeptide I was given.

In FIG. 8, the survival ratio (%) is plotted as ordinate vs. groups A toF which were each given the following amount of the pseudomonas asabscisso.

    ______________________________________                                        Group      Number of cells/mouse                                              ______________________________________                                        A          19,000                                                             B          3,800                                                              C          750                                                                D          150                                                                E           30                                                                F           6                                                                 ______________________________________                                    

(7) Testing IL-1α for Effect to Promote CSF Production

The following test was conducted using human embryonic lung fibroblasts(HFL-1, ATCC registered cell strain No. CCL-153) which produce CSF.

HFL-1 cells were suspended in hamster 12K culture (Ham, R.G., Proc.Natl. Acad. Sci., 53, 288 (1965) containing 10% FCS to a concentrationof 2×10⁵ cells/ml.

IL-1α obtained in Example 1 was added to portions of the cell suspensionin varying concentrations. Each of the mixtures was incubated in acarbon dioxide gas incubator at 37° C. for 24, 48 or 72 hours. Theculture supernatant was collected, and the amount of CSF produced andaccumulated in the supernatant was measured using mouse bone marrowcells (Lewis, I.C. et al, J. Immunol., 128, 168 (1982)).

The results are listed in Table 3 below.

                  TABLE 3                                                         ______________________________________                                        Concentration of incubated IL-1α (GIF units/ml)                         Time     0            1       10                                              (day)    CSF activity (U/ml)                                                  ______________________________________                                        1        0            190     130                                             2        0            240     145                                             3        0            100     120                                             ______________________________________                                    

The above results reveal that the addition of IL-1α promotes CSFproduction by HFL-1 cell strain.

(8) Method of Preparing Cytokines from Animal Cells

HSB-2 C5B2 cells (J. Immunol., 131, 1682-1689 (1985)), in an amount of2×10⁵ cells/well, were incubated for 24 hours in the presence of varyingconcentrations of polypeptide II and 0.01% PHA-P. The IL-2 activity ofthe supernatant collected was measured by the method of K. A. Smith etal. using IL-2 dependent mouse T cells (CTLL 2) (J. Immunol., 120, 2027(1978)). Table 4 below shows the results.

                  TABLE 4                                                         ______________________________________                                        Concentration of                                                              polypeptide II                                                                              IL-2 activity (cpm × 10.sup.-3)                           (ng/ml)       mean (n = 5)                                                    ______________________________________                                        0             0.4                                                             0.0005        0.4                                                             0.005         0.6                                                             0.05          2.1                                                             0.5           3.2                                                             5             3.5                                                             50            3.9                                                             500           3.8                                                             ______________________________________                                    

These results indicate that the use of the polypeptide of the inventionpermits animal cells to produce natural-type cytokines efficiently.

The amount of the present polypeptide to be used for the above methodcan be very small, usually about 10 ng/ml, whereby satisfactory resultsare achievable, while the use of the polypeptide facilitatespurification of the cytokine derived.

(9) When cytokines are to be produced from animal cells, it is essentialthat the polylpeptide of the invention used for inducing the productionbe stable in structure under the prevailing condition and be bound tothe IL-1 receptor on the surface of the cells. More specifically, it isrequired that the present polypeptide bind to the IL-1 receptor andtransmit to the cell a signal necesary for the production of thecytokine.

Accordingly, the following test was conducted in connection with thebinding of the present polypeptide to the IL-1 receptor on fibroblasts.

Balb/3T3 cells (clone A31: ATCC, CCL-163, 1×10⁶ cells/well) almostuniformly grown over a 6-well plate were reacted at 4° C. for 2 hourswith 50000 cpm/well of ¹²⁵ I-labelled IL-1β (Seikagaku (Biochemistry)58, No.8, p. 840 (1986); EPO No.187991) and 20 ng/ml of IL-1β orpolypeptide II preincubated at 37° C. for 24 hours in D-MEM supplementedwith 10% FCS. The liquid reaction mixture was discarded with a Pasteurpipette, the cells were gently washed with 1 ml of D-MEM supplementedwith 10% FCS, and the supernatant was discarded. After repeating thewashing procedure twice, the cells were solubilized with 1 ml of amixture of 1% SDS and 0.2 N NaOH. The radioactivity (boundradioactivity) of the solubilized cell solution and the liquid used forwashing the wells was measured by a gamma-counter.

The ¹²⁵ I-labeled IL-1β used was prepared and purified by the method ofBolton and Hunter (Biochem. J., 133, 529 (1973)). Specific activity: atleast 250 μCi/μg protein.

Table 5 shows the results.

                  TABLE 5                                                         ______________________________________                                                    Inhibition activity (%)                                           ______________________________________                                        Polypeptide II                                                                              100                                                             IL-1β    About 4                                                         ______________________________________                                         ##STR3##                                                                     -   wherein                                                               

A: Bound radioactivity in absence of unlabelled polypeptide I

B: Experimental value of bound radiocativity

C: Radioactivity unspecifically absorbed on the plate

This index represents the binding activity of the conjointly presentIL-1β or polypeptide II on the IL-1 receptor. Incidentally, it is knownthat IL-β and IL-1β are in common in respect of the IL-1 receptor.

Table 5 reveals that polypeptide II retains the satisfactory bindingactivity on IL-1 receptor even under cytokine induction-productionconditions and is therefore useful for such application.

PREPARATION EXAMPLE 1

To a solution of 1×10⁷ units/ml, calculated as GIF activity, ofpolypeptide I in saline was added human serum albumin (HSA) to aconcentration of 0.5%. The mixture was filtered (0.22 μm membranefilter), placed into vials, 1 ml, in each, in sterile state, andlyophilized to obtain a preparation for injection.

The preparation is used as dissolved in 1 ml of distilled water forinjection.

We claim:
 1. A method for the treatment of a pateint afflicted witharthritis or inflammation comprising administering to said patient apharmaceutically effective amount of interleukin-1α.
 2. The methodaccording to claim 1, wherein said interleukin-1α is humaninterleukin-1α.
 3. The method according to claim 1, wherein saidinterleukin-1α is administered by injection.
 4. A method for thetreatment of a patient afflicted with inflammation, comprisingadministering to said patient a pharmaceutically effective amount ofinterleukin-1α.
 5. The method according to claim 4, wherein saidinterleukin-1α is human interleukin-1α.
 6. The method according to claim4, wherein said interleukin-1α is administered by injection.